The long-term goal of this proposal is to elucidate how a heterooligomeric protein folds and assembles in the lumen of endoplasmic reticulum(ER), and how the cell controls the quality of exported proteins in the secretory pathway. The protein to be analyzed is LFA-1, a lymphocyte specific integrin, which plays a crucial role in cell-cell interactions during inflammation. A defect in folding and assembly of this heterodimeric membrane glycoprotein results in a human genetic disease called leukocyte adhesion deficiency. Better understanding of its folding and assembly should enhance our ability to control the cellular reactions during inflammation, and to understand and treat the disease. The specific aims of this proposal are: (1) To identify any N-terminal fragments of LFA-1 that are correctly folded and secretion-competent by analyzing the secreted polypeptides from LFA-1 transfected cells in the presence of low concentrations of puromycin. Puromycin is a protein synthesis inhibitor that creates random C-terminal truncations by premature chain termination; (2) To define the criteria that cells use to control the quality of exported LFA-1 by analyzing folding and assembly of different fragments of LFA-1 as well as individual alpha and beta chains and the intact dimers. (3) To identify the molecular chaperones interacting with LFA-1 in the ER and in the secretory pathway by using a novel site-specific photo-crosslinking method. The results should not only provide insight into the folding, assembly and quality control processes of LFA-1, but provide a basis for production of folded soluble fragments and thus facilitate its structural and functional analysis.